PBMC medium

• 500 mL RPMI1640
  + 5 mL 100× L-glutamine stock solution (2 mM final concentration)
  + 25 mL human AB serum (5% final concentration)
  

Reconstitution of PepTivators

1. For reconstitution of the lyophilized peptide pool take the vial from –20 °C and warm-up to room temperature.
   ▲ Note: Do not open the vial by removing the rubber plug.
2. To dissolve the 6 nmol version of the PepTivator, fill a sterile syringe (0.5 mL) with 200 μL of sterile water. To dissolve the 60 nmol version of the PepTivator, fill a sterile syringe (5 mL) with 2 mL of sterile water.
3. Slowly inject the water with a sterile needle through the center of the rubber plug into the vial containing the lyophilized peptide pool.
4. Vortex the solution to completely dissolve the lyophilized peptide pool. The concentration of the stock solution of PepTivator Peptides is 30 nmol (approximately 50 μg) of each peptide per mL. 
5. Remove the rubber plug and aspirate the stock solution with a pipette.
6. To avoid repeated freeze-thaw cycles, prepare working aliquots from the stock solution.
7. Store the working aliquots at –80 °C. 

1. Perform a sterile preparation of PBMCs from fresh buffy coats or whole blood using Ficoll-Paque™.
   ▲ Note: The following may be a useful protocol:
   Isolation of mononuclear cells from human peripheral blood by density gradient centrifugation
   ▲ Note: To remove platelets after density gradient separation, resuspend cell pellet in buffer and centrifuge at 200×g for 10−15 minutes at 20 °C. Carefully aspirate supernatant. Repeat washing step.
   
2. Proceed to "in vitro stimulation for induction of CD154 expression".

1. Resuspend cells at a density of 1×10⁷ cells/mL in culture medium RPMI 1640 with L-glutamine and 5% human AB serum.
2. For each non-stimulated control and antigen-stimulated sample transfer 1×10⁷ cells to one well of a 24-well plate.
   ▲ Note: With the Gas-permeable Culture Plate # 150-000-362, up to 2.5×10⁷ PBMCs/well/mL can be stimulated as opposed to 1×10⁷ PBMCs/well/mL in standard 24-well plates.
3. Add 10 μL of CD40 pure – functional grade (1 μg/mL; 1:100) to each well.
4. Add antigen according to manufacturer’s recommendation to the antigen-stimulated wells, but not to control wells. When using PepTivators: Add 20µl of reconstituted stock solution per well.
5. Incubate cells for 4 hours at 37 °C, 5% CO₂.
6. Add brefeldin A to a final concentration of 2 µg/mL.
7. Incubate cells for another 2 hours at 37 °C, 5% CO₂.

Buffer (standard wash and dilution buffer)

• Prepare a solution of PBS, pH 7.2, 2 mM EDTA and 0.5% BSA by diluting MACS® BSA Stock Solution 1:20 with autoMACS^® Rinsing Solution. 

Antibody staining mix

Freshly prepare 200 μL of antibody staining mix for each well:

• 12.5 μL CD3-VioBlue®
• 25 μL CD4-APC
• 25 μL CD8-PerCP
• 25 μL CD14-PerCP
• 50 μL CD20-PerCP
• 25 μL CD154-Biotin(CD154 MicroBead Kit)
• 37.5 μL FcR Blocking Reagent
  

1. Centrifuge plate for 5 minutes at 300×g at room temperature.
2. Very carefully aspirate 800 μL of cell culture supernatant. Avoid resuspension of the cells.
3. Add 200 μL of antibody staining mix to each well (see above)
   
4. Mix well for 2 minutes using an orbital shaker.
5. Incubate for 3 minutes in the dark at room temperature.
6. Add 20 μL of Anti-Biotin MicroBeads UltraPure (from CD154 MicroBead Kit) to each well. Mix well for 2 minutes using an orbital shaker.
7. Incubate for 13 minutes in the dark at room temperature.
8. Add 300 μL of Inside Fix (from Inside Stain Kit) to each well. Mix well for 2 minutes using an orbital shaker.
9. Incubate for 13 minutes in the dark at room temperature.
10. Add 1 mL of buffer to each well and mix well.
11. (Optional) Take an aliquot of 200 μL cell suspension for staining of the original fraction.
    

1. Place MS Column in the magnetic field of a suitable MACS® Separator.
2. Prepare MS Column by rinsing with 500 μL of buffer.
3. Apply cell suspension onto the column.
4. Wash cells by rinsing the column with 500 μL of buffer, followed by 2×500 μL of Inside Perm (from Inside Stain Kit).
5. Prepare a solution of 10 μL of CD154-FITC and 90 μL of Inside Perm (from Inside Stain Kit).
6. (Optional) Add additional staining antibodies to the solution, e.g., for the staining of cell surface antigens internalized upon cell activation or antigens which accumulate in the cell.
   ▲ Note: Do not exceed the total volume of 150 μL.
7. Apply the solution onto the column and incubate for 10 minutes at room temperature.
   ▲ Note: The MACS Column has a flow-stop mechanism that will retain the solution in the column.
8. Wash cells by rinsing the column with 2×500 μL of Inside Perm (from Inside Stain Kit) followed by 500 μL of buffer.
9. Remove column from the separator and place it on a suitable collection tube.
10. Pipette 500 μL of buffer onto the column.
11. Cells are now ready for analysis. Store cells at 2–8 °C in the dark until analysis. Mix well before flow cytometry acquisition.
    ▲ Note: Samples may be stored at 2–8 °C in the dark for up to 24 hours.
    ▲ Note: Do not use propidium iodide (PI) or 7-AAD staining in general.

1. Wash cells by adding 1 mL of buffer. Centrifuge cells for 5 minutes at 300×g at room temperature.
2.  Aspirate supernatant.
3. Resuspend cells in 500 μL of Inside Perm (from Inside Stain Kit) vortex well. Centrifuge cells for 5 minutes at 300×g.
4. Aspirate supernatant.
5. Prepare a solution of 10 μL of CD154-FITC and 90 μL of Inside Perm (from Inside Stain Kit).
6. (Optional) Add additional staining antibodies to the solution, e.g., for the staining of cell surface antigens internalized upon cell activation or antigens which accumulate in the cell.
   ▲ Note: Do not exceed the total volume of 150 μL.
7. Add staining solution to the cells and incubate for 10 minutes at room temperature.
8. Wash cells by adding 1 mL of Inside Perm (from Inside Stain Kit). Centrifuge cells for 5 minutes at 300×g.
9. Aspirate supernatant.
10. Resuspend cells in 250 μL of buffer.
11. Samples are ready for measurement now.
    ▲ Note: Samples may be stored at 2–8 °C in the dark for up to 24 hours.

1. Bacher, P. et al. (2013) J. Immunol. 190: 3967–3976.
2. Bacher, P. and Scheffold, A. (2013) Cytometry A 83: 692–701.
   

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